1. Field of the Invention
The invention is generally directed to the field of catalytic antibodies, in particular antibodies catalyzing the deamidation of proteins, and haptens for the induction and/or screening of such antibodies, and the use of such antibodies to catalyze the deamidation of proteins.
2. Background of the Invention
Advances in the fields of catalytic chemistry and immunochemistry have recently led to the tapping of the immune system to produce antibodies with a specific important function, catalysis of specific chemical reactions. This new technology provides the potential to customize highly selective catalysts for potential uses in various fields of biology, chemistry and medicine.
A fundamental concept in catalysis is the transition-state theory. According to this theory, a chemical reaction can be visualized in terms of the free energy changes of the reactants as a function of a reaction coordinate. The reaction coordinate having the highest free energy is the most unstable chemical species, termed the "transition state". Enzymes are now understood to accelerate chemical reactions by enlisting their binding energy to stabilize activated species undergoing chemical transformation, by which manner enzymes provide a pathway of lower free energy for the reaction mechanism.
Meanwhile, advances in immunochemistry have led to a better understanding of the nature of antibodies, and methods for the production of specifically desired antibodies. Antibodies are large proteins consisting of four polypeptide chains: specifically two identical heavy chains and two identical light chains. The light chains are divided into two domains, variable (V.sub.L) and constant (C.sub.L), while the heavy chains consist of four domains (V.sub.H, C.sub.H1, C.sub.H2, and C.sub.H3). The structure of an antibody is shown schematically in FIG. 1. Antibodies bind to ligands by means of a combining site comprised of a hypervariable region which consists of six loops of extended chains, three each from the light- and heavy-chain variable domains. The hypervariability of these regions provides the immune system with the ability to generate a large number of ligand-specific antibodies having a variety of binding characteristics.
About forty years ago, it was first pointed out by Pauling that the fundamental processes which determine binding of enzymes and antibodies are the same. Both achieve binding by the use of ordinary forces which occur when small molecules come within a few angstroms of each other. Of course, there are specific differences between enzymes and antibodies, but the similarity in binding specificity has led to the advance of catalytic antibodies as specific compounds for catalysis.
The potential of the immune system to perform chemistry was clearly recognized in 1986 when Schultz and Lerner first showed that antibodies raised to tetrahedral, negatively-charged phosphate and phosphinate transition state analogues could selectively catalyze the hydrolysis of carbonates and esters, respectively. Phosphinate and phosphonamidate analogues are thought to closely mimic the transition states for ester and amide hydrolysis, respectively, and have been used to design antigens for production of catalytic antibodies.
Since that time, antibodies have been generated which catalyze a wide variety of chemical reactions ranging from paracyclic to peptide bond cleavage, and including specifically antibodies which catalyze carbonate hydrolysis, ester hydrolysis, amide hydrolysis, Claisen rearrangement, amide bond formation, lactonization, transesterification and photo-induced cleavage (see for example Lerner, et al., Science 252:659 (1991) hereby incorporated by reference). The specificity of such antibody-catalyzed reactions has been shown to equal or even exceed that of the corresponding enzymatic reactions.
One of the challenges in this field is the design of an appropriate antigen which can mimic the transition state compound of the reaction of interest. The antigen must, of course, first induce antibodies which can catalyze the reaction of interest. But the antigen must induce antibodies which have a greater stabilizing interaction with reaction intermediates than with either the reactant or product in order to avoid non-catalytic stoichiometric complexes. Enzymes are particularly adapted to exert precise stereo-chemical control over the reactions which they catalyze. Antibodies, being ligand (antigen) specific, should also be able to catalyze stereo-selective reactions, provided proper attention is successfully paid to the symmetry between antigens and substrates. Early attempts to prepare catalytic antibodies were not successful, apparently in large part due to a failure to properly address the mechanistic requirements of the chemical transformation under study.
An important recent advance in immunology involves the preparation of monoclonal antibodies, for example as described by Kohler and Milstein (Nature 256:495 (1975)). These authors demonstrated that it was possible to generate monoclonal antibodies which consist of a single distinct molecular structure, thereby generating large amounts of homogeneous antibodies with desired specificity. According to this procedure, after an immunogenic response to a desired immunogen is achieved, antibody-producing plasma cells from, for example, the spleen are fused or hybridized to an immortal myeloma cell line. This enables the antibody-producing cells to be cultured in vitro indefinitely. Hybridomas can then be cloned and separated into colonies which produce a single antibody, With the resulting cells being screened for their ability to generate antibodies with the desired specificity and high affinity to the ligand of interest.
Immunogenic responses in the preparation of monoclonal antibodies have also been successfully accomplished with haptens. Haptens are small molecules which are not themselves immunogenic. Immunogenic response, however, can be stimulated if the haptens are coupled to an antigenic carrier molecule. Various coupling molecules can be utilized as known in the art, but particularly preferred molecules for use as carriers are the proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Haptens coupled to BSA and KLH stimulate the production of monoclonal antibodies having specificity for the hapten of interest.
As a result, one approach which has developed for generating antibodies that catalyze a specific class of reactions is by synthesis of a hapten structure which mimics the transition state structure of the reaction to be catalyzed. A first experimental demonstration of this notion related to acyl transfer reactions, specifically simple hydrolytic reactions. Relatively stable phosphate, phosphinate and phosphonamide species were prepared to provide the transition state analogues, and antibody specific for the transition state analogues acted as catalysts with rate accelerations on the order of 10.sup.3 to 10.sup.4 over uncatalyzed reactions (See for example, Lerner et al., above).
There are now some 50 reactions that are known to be catalyzed by antibodies. At least conceptually, the most susceptible reactions to antibody catalysis would be a class of reactions, originally viewed as "no mechanism" reactions. Claisen rearrangement and Diels-Alder addition are two such examples.
One additional specific reaction receiving recent attention is deamidation. Deamidation of proteins is a process wherein the gamma-amino groups of asparagine or glutamine are lost to produce aspartic acid and glutamic acid, respectively. Generally, deamidation has profound affects of the folding of a protein and is a principle cause of irreversible denaturation. In some cases deamidation increases susceptibility of the protein to proteolytic degradation. Non-enzymatic deamidation is a slow process under physiological conditions. In the cases where the rate has been measured, the half-time of the reaction ranges from 8 to 80 days for proteins and from 6 to 277 days for peptides. Robinson and Rudd (Curr. Top. Cell. Regul., 8:248 (1974)) reviewed peptide and protein deamidation, and discussed possible roles for this reaction, in vivo. Subsequent research suggests that deamidation of aspargine (Asn) and glutamine (Gln) may be more widespread than initially thought. The existence of an enzyme which recognizes and modifies one of the products of non-enzymatic deamidation supports proposals that this post-translational modification plays a role in physiological processes. Deamidation is fundamentally a hydrolytic reaction, formally similar to the peptide-bond cleavage reaction, which is catalyzed by proteases. A schematic representation the mechanism for acid- and base-catalyzed deamidation reactions is shown below: ##STR1## The general acid (HA) catalyzes the reaction by protonating the amido NH.sub.2 -leaving group of the ASN side chain. A general base can attack the carbonyl carbon of the amido group or activate another nucleophile by abstraction of a proton for attack on the amide carbon. Deamidation has been studied for a variety of specific proteins, a review of which is set forth in Wright, Critical Reviews in Biochemistry and Molecular Biology, 26:1 (1991), which is hereby incorporated by reference. These specific proteins include at least the following:
Aspartate aminotransferase PA0 Calmodulin PA0 Carbonic anhydrase (B, C) PA0 Cytochrome C (horse heart) PA0 Dihydrofolate reductase (recombinant) PA0 Glucagon PA0 Hemoglobin Providence (human) PA0 Hemoglobin Singapore (human) PA0 Hemoglobin Wayne (human) PA0 Insulin (human, bovine) PA0 Lysozyme PA0 Ribonuclease A (bovine) PA0 Ribonuclease (bovine seminal) PA0 Trisephosphate isomerase (human) PA0 Trypsin (bovine) PA0 Trypsin inhibitor (bovine) (human) PA0 Tryptophan synthase PA0 Adrenocorticotropin (porcine, ovine, human) PA0 Alcohol dehydrogenase (Drosophila) PA0 Aldolase (rabbit muscle) PA0 Amyloid serum protein (human) PA0 Calbindin (recombinant) PA0 Chloroperoxidase (Caldariomyces fumago) PA0 Cholera toxin B chain PA0 Crystallin.alpha.A (human) PA0 Crystallin.alpha.A (chicken) PA0 Crystallin.alpha.B.sub.2 (bovine) PA0 Crystallin.beta.B.sub.p (bovine) PA0 Crystallin.epsilon.(duck) PA0 Epidermal growth factor PA0 F.sub.2 coat protein PA0 Growth hormone (human) PA0 Growth hormone (bovine) PA0 Histone H4 (human) PA0 Hypoxanthine-guanine phosphoribosyltransferase PA0 Immunoglobulin .lambda. chain (mouse) PA0 Interleukin.alpha.1 (human) PA0 Myelin basic protein (bovine) PA0 Neocarzinostatin PA0 Ovalbumin PA0 Parathyroid hormone (human) PA0 Prolactin (ovine, bovine) PA0 Ribonuclease U.sub.2 (Ustilago sphaerogena) PA0 Serine hydroxymethyltransferase PA0 Somatotropin (human) PA0 Substance P (human) PA0 Acetylcholinesterase (cobra venom) PA0 Amylase (human salivary) PA0 Enterotoxin B (staphylococcus) PA0 Phosphoryl carrier protein PA0 Proteinass (alkaline)
It has been shown that the irreversible thermal denaturation of ribonuclease, lysozyme and .alpha.-amylase is controlled largely by the deamidation of Asn and Gln at acid and neutral pH and that a single deamidation in ribonuclease can affect folding kinetics. The observation that deamidation of Asn residues is a principle cause for the irreversible denaturation of proteins is an important aspect which implicates the role of Asn and Gln and the folding of proteins, their assembly into biologically active complexes, and their breakdown.
There are instances where one may wish to inactivate specific proteins using a deamidation process to promote unfolding of the target polypeptide. One example of such would be the selective disruption of a coat protein of a virus so as to prevent binding of the virus to its cellular receptor, thus preventing infection of the cell by the virus. Another example would be the disruption of the adhesion molecules on the surface of platelet cells, by which the platelets begin the process of blood clotting, in order to prevent clot formation. In such cases, it is clearly necessary to greatly accelerate the deamidation reaction. Catalysis of the reaction by an enzyme is a means of increasing the rate of deamidation under physiologically conditions.
The present invention, therefore, is focused on the discovery of an antibody which is capable of catalyzing a deamidation reaction, specifically the deamidation of an asparaginyl-glycyl dipeptide, which dipeptide may be contained in a larger polypeptide.